Increased protein stability of CDKN1C causes a gain-of-function phenotype in patients with IMAGe syndrome.

Mutations in the proliferating cell nuclear antigen (PCNA)-binding domain of the CDKN1C gene were recently identified in patients with IMAGe syndrome. However, loss of PCNA binding and suppression of CDKN1C monoubiquitination by IMAGe-associated mutations hardly explain the reduced-growth phenotype characteristic of IMAGe syndrome. We demonstrate here that IMAGe-associated mutations in the CDKN1C gene dramatically increased the protein stability. We identified a novel heterozygous mutation, c.815T>G (p.Ile272Ser), in the CDKN1C gene in three siblings manifesting clinical symptoms associated with IMAGe syndrome and their mother (unaffected carrier). PCNA binding to CDKN1C was disrupted in the case of p.Ile272Ser, and for two other IMAGe-associated mutations, p.Asp274Asn and p.Phe276Val. Intriguingly, the IMAGe-associated mutant CDKN1C proteins were fairly stable even in the presence of cycloheximide, whereas the wild-type protein was almost completely degraded via the proteasome pathway, as shown by the lack of degradation with addition of a proteasome inhibitor, MG132. These results thus suggested that the reduced-growth phenotype of IMAGe syndrome derives from CDKN1C gain-of-function due to IMAGe-associated mutations driving increased protein stability.